Figure 5

Lipid raft disruption inhibits the levels of pSrc, pFAK, pCav, pERK, pAkt, pPI3-K, and pAkt in breast carcinoma cell lines. (A) and (B) MDA-MB-231 and ZR 751 cells left untreated or pretreated with different concentrations of MβCD were subjected to Western blot analysis for total and phosphorylated forms of Src, FAK, Cav, ERK, PI3-K, and Akt. GAPDH was used to verify that similar amounts of protein were loaded in each lane. Densitometric analysis of phosphorylated molecules was done. The result provided is of a representative experiment repeated 3-4 times with concordant results (*p < 0.01, **p < 0.001). (C) MDA-MB-231 cells were left untreated or pretreated with 7.5 mM MβCD for 1 hr at 37°C, washed, labeled with primary antibodies to GM1 by Vybrant lipid raft labeling kit, fixed, permeabilized, incubated with pFAK, pCav and pPI3-K antibodies and appropriate secondary antibodies. The cells were washed, stained with DAPI containing anti-fade agent, and visualized by confocal immunofluorescence microscopy.